Evaluation of the antimicrobial effects of MTAD, NaOCl against selected endodontic pathogens.

INTRODUCTION
The aim of this study was to compare the antimicrobial effects of MTAD, sodium hypochlorite (NaOCl) and their combination on endodontic micro-organisms.


MATERIALS AND METHODS
Zone of Inhibition (ZI) and Minimum Inhibitory Concentration (MIC) were the techniques used. In ZI technique blood agar plates were inoculated with organisms, paper discs were soaked with irrigants and maximum zones of bacterial inhibition were recorded. In the MIC technique the irrigants were serially diluted in TSB tubes and 0.1 mL of the tested microbe solutions were added. Results were obtained on the basis of turbidity and growth on agar plates. Statistical analyses were carried out using ANOVA and Tukey tests.


RESULTS
In ZI technique, we investigated 120 specimens including 5 microbial species, 3 irrigants and their control groups, each with 6 repetitions. The results demonstrated MTAD greater antimicrobial efficacy compared to NaOCl, and their mixture (M+N) against Staphylococcus (S) aureus, Enteric (E) bacteria and Enterococcus (E) faecalis (P<0.001). NaOCl was more effective in eradicating Candida (C) albicans than the others (P<0.01). MIC method (155 tubes) showed MTAD to be more effective against E. bacteria and S. aureus. MTAD and NaOCl were equally effective against E. faecalis; however, NaOCl was more effective against C. albicans.


CONCLUSION
Bacterial species were more susceptible to MTAD than NaOCl, C. albicans, however, was more susceptible to NaOCl. The advantage of NaOCl is that it has broad spectrum antimicrobial activity. The joint solution (M+N) did not prove to be more effective than their individual use.


INTRODUCTION
It is well established that pulp and periapical disease as well as failed root canal therapy (RCT) are due to the presence of microbes in the root canal system (1). Eliminating microbes from the infected root canals and prevention of re-infection are one of the fundemental aims of RCT. Clinical investigations have shown that chemo-mechanical preparation with the use of anti microbial medicaments will effectively reduce the microbial content of the canals (2)(3)(4). Despite these meticulous efforts some micro-organisms stay within the canal (4-7). In a clinical study evaluating the effect of infection on the RCT outcome, Sjogren et al. cultured samples from the root canals after preparation and before obturation. When the cultures were negative, the success rate was 94%; when bacteria were found however, success rate was reduced to 68% after 5 year follow up (5). Extensive research has discovered various microorganisms in root canal failures (Chronic Apical Periodontitis) such as Enterococci (8- 15), Actinomyces (13), Streptococci (13), Asna Ashari et al.

MATERIALS AND METHODS
The investigation was carried out in Shahid Beheshti University Dental School and approved by the ethical committee. The microbes tested were E. faecalis, Pseudomonas (P) aeruginosa, Escherichia (E) coli, S. aureus and C. albicans. E. faecalis was incubated in Bile Scoline Agar, P. aeruginosa and S. aureus in Blood Agar, E. coli in Esoin Methylen Blue (EMB), and C. albicans in Sabouraud Dextrose Agar plates.
After 24 hours a suspension was prepared from each microbial species with a concentration of 1 McFarland (i.e. 3×10 8 Colony of bacteria in the Trypticase Soy Broth environment) and used in the investigation. To assess the antimicrobial power of each irrigant ZI and MIC was employed. In the ZI technique 30 blood agar plates (batch no: 108860500, Merck KGaA, Darmstadt, Germany) were prepared according to manufacture's instructions and the 125 mm Petri dishes were uniformly covered. These were then stored for 2 days in 37°C to make sure sterilized conditions were met and bacteria were not introduced. The sterile paper discs (batch no: 1288502, Padtan Teb, Tehran, Iran) diameter of 6 mm were divided into three groups. Each group was soaked either with 4 drops (0.2 mL) of NaOCl, MTAD or an equal mixture of MTAD and NaOCl (M+N). Five mL from each 1 McFarland suspension was removed with a sterile swab next to a flame and under the ventilation hood and placed on a blood agar plate in a criss-cross fashion. Each culture area was then divided into 4 sections in the following manner: the first section contained disc soaked with BioPure MTAD (batch no: 050805, Dentsply Tulsa Dental, Tulsa, OK), the second section with NaOCl 5.25% (batch no: 28095, Pure Sodium Hypochlorite, Chemeen Company, Iran) and the third section contained disc soaked with equal amounts of MTAD and NaOCl (5.25%), the final section consisted of normal saline (batch no: 173885 A, 0.9% Sodium Chloride, Medicines and Drugs Company, Iran). This was repeated 6 times for each of the microbes; altogether 120 samples were taken. Samples were placed in the incubator (batch no: 0323212100, Farateb Tajheez Specialist Company, Iran) for 48 hours at 37°C and 10% CO 2 . The maximum microbial ZI around each disc was then measured and recorded in millimetres. In the MIC technique 30 test tubes were prepared for each microbe. One mL of Trypticase Soy Broth (TSB) (batch no: 105459, Merck KgaA, Darmstadt, Germany) was poured into each test tube and then autoclaved (sterilization). The tubes were then divided into three groups of 10 test tubes each. One mL of MTAD and MTAD+NaOCl were partially opaque and therefore a culturing method was also employed. After measuring turbidity of the test tubes, 0.5 cc of each tube solution was placed on Nutrient Agar (batch no: 1054430500, Merck KGaA, Darmstadt, Germany) and placed in the incubator for 48 hours. One hundred and fifty five test tubes were used to test the 5 individual microbes, 5 of which were controls. The MIC of the irrigants for each individual microbe was recorded based on the degree of turbidity (with the naked eye) and the absence of microbial growth on the agar plates. The experiment was carried out in accordance with sterilisation principles (45). The relevant results of ZI were added into SPSS for Windows V 11.5. Descriptive criteria (central values, measurements of dispersion and confidence interval levels) were calculated and the data was analysed with ANOVA and Tukey tests and P<0.05 was considered statistically significant.

Asna Ashari et al.
MIC results illustrated that MTAD and NaOCl were strong antimicrobials and may be equally effective even when diluted. In this study MTAD was more effective against S. aureus and E. bacteria, however, NaOCl 5.25% and MTAD were equally effective against E. faecalis (Table 4).

DISCUSSION
The results of this in vitro investigation reiterate the superiority of MTAD compared to NaOCl and M+N against S. aureus, E. bacteria and E. faecalis by the ZI method. The poorest antimicrobial agent was shown to be the mixture of M+N. Experiments carried out by Davis et al. (40) and Krause et al. (43) in 2007 both proved MTAD to be more effective against E. faecalis than NaOCl 5.25%. This study also demonstrates that MTAD is effective against a range of bacteria. Tay et al. carried out a similar study that compared MTAD, NaOCl and the consecutive use of the irrigants (instead of combination) (46). They used a concentration of 1.3% NaOCl; their results confirmed that MTAD was the most effective irrigant in eliminating E. faecalis. However, they found no difference between NaOCl and N+M; possibly due to the different concentration of NaOCl, bacterial species and/or slightly different incubation conditions employed. Torabinejad et al. has compared the effectiveness of MTAD and NaOCl (5.25%) using the ZI technique and discovered their similar antibacterial action against E. faecalis (38). However, in this study after dilution MTAD was shown to have a significantly greater ZI than NaOCl. Interestingly the antimicrobial effect of the mixture (M+N) was less than MTAD or NaOCl alone. A recent study showed that MTAD used alone has greater substantivity than other irrigants (47). NaOCl reduces the antimicrobial power of MTAD; both irrigants are stronger antimicrobials when used independently. Attention must be paid to the oxidation of MTAD by NaOCl which reduces the substantivity and antimicrobial power of MTAD; similar to the peroxidation of tetracycline with Reactive Oxygen Species (ROS), confirmed in Tay's study (46). There have been reports that antioxidant such as ascorbic acid rinse following NaOCl irrigation will remove remnants of hypochlorite (48). C. albicans distorts the findings as NaOCl is stronger agent than MTAD and M+N; MTAD showed no antifungal effects. Ruff et al's study also supports our results; they demonstrated that Chlorohexidine 2% and NaOCl 6% were most effective in reducing the CFU of C. albicans (49). There is a large body of evidence including this current study that find bacteria such as E. faecalis, strands of E. bacteria (P. aeruginosa, E. coli) and types of C. albicans to have greater resistance to irrigation when compared to S. aureus (8-21). These microorganisms have also been known to be resistant to antibacterials and intracanal medicaments such as calcium hydroxide (50)(51)(52). MIC results illustrated that MTAD and NaOCl were strong antimicrobials even after dilution. In this study, MTAD was more effective against S. aureus and E. bacteria, thought NaOCl 5.25% and MTAD were equally effective against E. faecalis. A similar study using MIC, Torabinejad et al. demonstrated that MTAD was effective when diluted 200 times, and NaOCl (5.25%) was effective when diluted 32 times against E. faecalis (38), concurring with our results (MTAD was found effective when diluted 32 times). This may be due to the various strands of E. faecalis, different amounts of doxycycline in MTAD (43), conditions of incubation used. The present study used Bio Pure MTAD (Dentsply, Tulsa Dental, Tulsa, OK) with 150 mg of Doxycycline i.e. 3%. MTAD is naturally opaque and therefore measuring its turbidity may cause discrepancy in the results. MTAD instructions require NaOCl to be used as the final irrigant (36). The results in this study and Tay et al's indicate that an interaction may occur altering the antimicrobial efficacy of MTAD (46). The advocacy of using these irrigants consequetively may be questioned.

Effect of intra canal medicaments
We suggest further investigations using irrigants in a polymicrobial environment to mirror endodontics infections.

CONCLUSION
MTAD is ineffective against C. albicans and its substantivity may be altered when used in conjunction with NaOCl. C. albicans is often present in resistant and secondary endodontic infections as well as in peri-radicular lesions. NaOCl is an inexpensive and readily available chemical with broad spectrum action which has stood the test of time.